The invention relates to biochemistry, cell biology, and in vivo optical imaging.
Optically based biomedical imaging techniques have advanced over the past decade due to factors including developments in laser technology, sophisticated reconstruction algorithms and imaging software originally developed for non-optical, tomographic imaging modes such as CT and MRI. Visible wavelengths are used for optical imaging of surface structures by means of endoscopy and microscopy.
Near infrared wavelengths (approx. 700-1000 nm) have been used in optical imaging of internal tissues, because near infrared radiation exhibits tissue penetration of up to 6-8 centimeters. See, e.g., Wyatt, 1997, xe2x80x9cCerebral oxygenation and haemodynamics in the fetus and newborn infant,xe2x80x9d Phil. Trans. R. Soc. London B 352:701-706; Tromberg et al., 1997, xe2x80x9cNon-invasive measurements of breast tissue optical properties using frequency-domain photo migration,xe2x80x9d Phil. Trans. R. Soc. London B 352:661-667.
Advantages of near infrared imaging over other currently used clinical imaging techniques include the following: potential for simultaneous use of multiple, distinguishable probes (important in molecular imaging); high temporal resolution (important in functional imaging); high spatial resolution (important in in vivo microscopy); and safety (no ionizing radiation).
In near infrared fluorescence imaging, filtered light or a laser with a defined bandwidth is used as a source of excitation light. The excitation light travels through body tissues. When it encounters a near infrared fluorescent molecule (xe2x80x9ccontrast agentxe2x80x9d), the excitation light is absorbed. The fluorescent molecule then emits light (fluorescence) spectrally distinguishable (slightly longer wavelength) from the excitation light. Despite good penetration of biological tissues by near infrared light, conventional near infrared fluorescence probes are subject to many of the same limitations encountered with other contrast agents, including low target/background ratios.
We have developed intramolecularly-quenched, near infrared fluorescence probes that emit substantial fluorescence only after interaction with a target tissue, i.e., xe2x80x9cactivation.xe2x80x9d This increases the target/background ratio by several orders of magnitude and enables non-invasive, near infrared imaging of internal target tissues in vivo, based on enzymatic activity present in the target tissue.
Accordingly, the invention features an intramolecularly-quenched fluorescence probe comprising a polymeric backbone and a plurality of near infrared fluorochromes covalently linked to the backbone at fluorescence-quenching interaction-permissive positions separable by enzymatic cleavage at fluorescence activation sites.
The backbone can be any biocompatible polymer. For example, it can be a polypeptide, a polysaccharide, a nucleic acid, or a synthetic polymer. Polypeptides useful as a backbone include, for example, polylysine, albumins, and antibodies. Poly(L-lysine) is a preferred polypeptide backbone. The backbone also can be a synthetic polymer such as polyglycolic acid, polylactic acid, poly(glycolic-colactic) acid, polydioxanone, polyvalerolactone, poly-xcex5-caprolactone, poly(3-hydroxybutyrate, poly(3-hydroxyvalerate) polytartronic acid, and poly(xcex2-malonic acid).
The probe can include one or more protective chains covalently linked to the backbone. Suitable protective chains include polyethylene glycol, methoxypolyethylene glycol, methoxypolypropylene glycol, copolymers of polyethylene glycol and methoxypolypropylene glycol, dextran, and polylactic-polyglycolic acid. In some embodiments of the invention, the backbone is polylysine and the protective chains are methoxypolyethylene glycol.
Fluorescence activation sites can be located in the backbone, e.g., when the fluorochromes linked directly to xcex5-amino groups of polylysine. Alternatively, each fluorochrome can be linked to the backbone by a spacer containing a fluorescence activation site. The spacers can be oligopeptides. Oligopeptide sequences useful as spacers include: Arg-Arg; Arg-Arg-Gly; Gly-Pro-Ile-Cys-Phe-Phe-Arg-Leu-Gly (SEQ ID NO:1); and His-Ser-Ser-Lys-Leu-Gln-Gly (SEQ ID NO:2).
Near infrared fluorochromes useful in this invention include Cy5.5, Cy5, Cy7, IRD41, IRD700, NIR-1, LaJolla Blue, indocyanine green (ICG) and analogs thereof, indotricarbocyanine (ITC), and chelated lanthanide compounds that display near infrared fluorescence. The fluorochromes can be covalently linked to the backbone, or spacers, using any suitable reactive group on the fluorochrome and a compatible functional group on the backbone or spacer. A probe according to this invention also can include a targeting moiety such as an antibody, antigen-binding antibody fragment, a receptor-binding polypeptide, or a receptor-binding polysaccharide.
The invention also features an in vivo optical imaging method. The method includes: (a) administering to a living animal or human an intramolecularly-quenched fluorescence probe that accumulates preferentially in a target tissue, and comprises a fluorochrome attachment moiety and a plurality of near infrared fluorochromes covalently linked to the fluorochrome attachment moiety at fluorescence-quenching interaction-permissive positions separable by enzymatic cleavage at fluorescence activation sites; (b) allowing time for (1) the probe to accumulate preferentially in the target tissue, and (2) enzymes in the target tissue to activate the probe by enzymatic cleavage at fluorescence activation sites, if the target tissue is present; (c) illuminating the target tissue with near infrared light of a wavelength absorbable by the fluorochromes; and (d) detecting fluorescence emitted by the fluorochromes. Preferably, the fluorochrome attachment moiety is a polymeric backbone. Alternatively, it can be a monomeric, dimeric, or oligomeric molecule.
The invention also features an in vivo optical imaging method comprising: (a) administering to a living animal or human an intramolecularly-quenched fluorescence probe comprising a fluorochrome attachment moiety and a plurality of near infrared fluorochromes covalently linked to the fluorochrome attachment moiety at fluorescence-quenching interaction-permissive positions separable by enzymatic cleavage at fluorescence activation sites, which enzymatic cleavage occurs preferentially in a target tissue; (b) allowing time for enzymes in the target tissue to activate the probe by enzymatic cleavage at fluorescence activation sites, if the target tissue is present; (c) illuminating the target tissue with near infrared light of a wavelength absorbable by the fluorochromes; and (d) detecting fluorescence emitted by the fluorochromes. Preferably, the fluorochrome attachment moiety is a polymeric backbone. Alternatively, it can be a monomeric, dimeric, or oligomeric molecule.
The above methods can be used, e.g., for in vivo imaging of a tumor in a human patient, or in vivo detection or evaluation of arthritis in a joint of a human patient The invention also features an in vivo method for selectively imaging two different cell or tissue types simultaneously. The method includes administering to an animal or human patient two different intramolecularly-quenched fluorescence probes, each of which accumulates preferentially in a target tissue. Each of the two probes includes a fluorochrome attachment moiety and a plurality of near infrared fluorochromes covalently linked to the fluorochrome attachment moiety at fluorescence-quenching interaction-permissive positions separable by enzymatic cleavage at fluorescence activation sites. Each of the two probes comprises a fluorochrome whose fluorescence wavelength is distinguishable from that of the other flurorochrome, and each of the two probes contains a different activation site.
As used herein, xe2x80x9cbackbonexe2x80x9d means a biocompatible polymer to which near infrared fluorochromes are covalently linked in fluorescence-quenching interaction-permissive positions.
As used herein, xe2x80x9cfluorescence activation sitexe2x80x9d means a covalent bond within a probe, which bond is: (1) cleavable by an enzyme present in a target tissue, and (2) located so that its cleavage liberates a fluorochrome from being held in a fluorescence-quenching interaction-permissive position.
As used herein, xe2x80x9cfluorescence-quenching interaction-permissive positionsxe2x80x9d means the positions of two or more atoms (in a single polymer) to which fluorochromes can be covalently linked (directly or indirectly through a spacer) so that the fluorochromes are maintained in a position relative to each other that permits them to interact photochemically and quench each other""s fluorescence.
As used herein, xe2x80x9cfluorochrome attachment moietyxe2x80x9d means a molecule to which two or more fluorochromes are covalently linked (directly or through a spacer) and maintained in fluorescence-quenching interaction-permissive positions relative to one another.
As used herein, xe2x80x9cprotective chainxe2x80x9d means a biocompatible polymeric moiety covalently linked to the backbone of a probe to inhibit undesired biodegradation, clearance, or immunogenicity of the backbone.
As used herein, xe2x80x9ctargeting moietyxe2x80x9d means a moiety bound covalently or noncovalently to a self-quenched probe, which moiety enhances the concentration of the probe in a target tissue relative to surrounding tissue.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application, including definitions will control. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference.
Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, preferred methods and materials are described below.. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the detailed description and from the claims.